This proposal is based on the hypothesis that progressive phospholipid degradation in energy-deficient myocytes alters membrane function, transport, and permeability, which initiates gross electrolyte and metabolic disturbances membrane damage leading to irreversible myocardial injury. Implicitly, some aspect in the maintenance of phospholipid metabolism within the cell must be impaired. Specific Aim 1 will study the role alterations in membrane phospholipid metabolism play in cellular damage. Specifically, how do metabolic changes, such as accelerated phospholipid degradation and impaired phospholipid synthesis, contribute to the progression of cell injury? Preliminary data indicate that decreased incorporation of fatty acids into cellular phospholipids accompanies increased de-esterification in a hypoxic myocyte culture model. Cellular phospholipase(s) must play a role in the degenerative process since progressive release of esterified fatty acids has been observed in most models. Specific Aim 2 will attempt to identify specific phospholipase(s) active in the elevated release of fatty acids from phospholipids. We have a unique advantage to pursue this goal; our recent studies have demonstrated a reduction in fatty acid release using the steroidal diamine U26,384. This putative phospholipase inhibitor will be utilized to screen phospholipase activities in cardiac myocytes to identify phospholipase(s) susceptible to inhibition by U26,384. Specific Aim 3 will determine whether the effects of phospholipase inhibitors are related to specific experimental models or whether their effects are consistent between the different models. In addition to the cellular systems utilized in Specific Aim 1, isolated heart perfusions will also be utilized as a model of ischemia. What initiates this imbalance during ischemia and hypoxia? Increases in cytosolic free calcium (Ca2+) occurring early in ischemic and hypoxic myocardial injury have been causally linked to the development of irreversible injury, due at least in part to increased membrane phospholipid degradation. Potential interactions between Ca2+ and phospholipid homeostasis will be addressed throughout these studies to evaluate correlation between phospholipid degradation, Ca2+ fluxes, and cell viability. Microspectrofluorometry of myocytes loaded with fura-2 and electron probe x-ray microanalysis will be used to measure free and total calcium levels, respectively. These studies seek to determine the specific changes in phospholipid metabolism underlying the pathogenesis of irreversible myocardial ischemic injury. Only by understanding these mechanisms can we develop methods to protect the ischemic myocardium from permanent damage.